Cloning of the SAP6 gene of Metschnikowia reukaufii and its heterologous expression and characterization in Escherichia coli.
The SAP6 gene (without signal sequence) encoding Metschnikowia reukaufii acid protease was amplified by PCR and fused to the expression vector pET-24a(+). The carboxy-terminal 6x His-tagged recombinant acid protease (rSAP6) was expressed from pET-24a(+)SAP6-6His in Escherichia coli BL21 (DE3) and purified with affinity chromatography using a Ni-NTA column. SDS-PAGE analysis and Western blotting revealed that the molecular mass of the purified rSAP6 was 54kDa. The optimal temperature and pH of the purified rSAP6 were 40 degrees C and 3.4, respectively. The enzyme was stable below 45 degrees C and between pH 2.6 and 5.0. The results show that Mn(2+) had an activating effect on the enzyme, while Cu(2+), Mg(2+), Zn(2+) and Ag(+) acted as inhibitors of the enzyme. However, Ca(2+) had no effect on the enzyme activity. The purified rSAP6 was characterized as an aspartic protease as it was inhibited by aspartic protease-specific inhibitors, such as pepstatin. It was also found that the purified rSAP6 had milk-clotting activity.
Li
Jing
J
UNESCO Chinese Center of Marine Biotechnology, Ocean University of China, Yushan Road, No. 5, Qingdao, Shandong 266003, China.
Chi
Zhenming
Z
Wang
Xianghong
X
D000595:Amino Acid Sequence; D000818:Animals; D057055:Aspartic Acid Proteases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D015153:Blotting, Western; D002413:Cations, Divalent / Q000494:pharmacology; D002846:Chromatography, Affinity / Q000379:methods; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D020536:Enzyme Activators / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D015870:Gene Expression; D006863:Hydrogen-Ion Concentration; D008670:Metals / Q000494:pharmacology; D055163:Metschnikowia / Q000201:enzymology* / Q000235:genetics; D008892:Milk / Q000378:metabolism; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010436:Pepstatins / Q000494:pharmacology; D016133:Polymerase Chain Reaction; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D016415:Sequence Alignment; D013696:Temperature
D002413:Cations, Divalent; D020536:Enzyme Activators; D004791:Enzyme Inhibitors; D005656:Fungal Proteins; D008670:Metals; D010436:Pepstatins; D011993:Recombinant Fusion Proteins; D057055:Aspartic Acid Proteases; C031375:pepstatin